RecA protein catalyzes homologous DNA pairing and strand exchange reactions that are the central processes of recombination and recombinational DNA repair in all cells. The structure of the RecA protein of Escherichia coli (Mr 37,842) features three distinct domains (32–34, the contents of which is incorporated by reference here in its entirety). The core domain (residues 31–269) includes the ATP and DNA binding sites. The core is flanked by smaller N- and C-terminal domains. The C-terminal domains (residues 270–352) appear as distinct lobes on the surface of RecA filaments, which shift position markedly in response to the presence of different bound nucleotides (35,36). The far C-terminus of the RecA protein (defined here as the C-terminal 25 amino acid residues) exhibits a preponderance of negatively charged amino acids, with seven Glu or Asp residues in the terminal 17 residues. This function of this region has been explored with the use of C-terminal deletions (37–42). Several of these studies documented the in vitro effects of deleting all or most of the C-terminal 25 amino acid residues. The major effects were an improvement of binding to dsDNA (37–39) and an evident alteration of the conformation of the core domain (42).
Currently a main function of RecA is strand exchange which is initiated when RecA binds to single-stranded DNA (ssDNA) within a gap or a ssDNA terminal extension. RecA protein binds DNA in at least two steps. The first is a slow nucleation step (1–3), and this is followed by a rapid, cooperative binding of additional monomers to lengthen the filament uniquely in a 5′ to 3′ direction (4,5). The resulting RecA-ssDNA complex has an extended, helical conformation, with approximately 6 RecA monomers and 18 nucleotides (nt) of DNA per right-handed helical turn (18.6 base pairs per turn in RecA filaments with bound ATPγS (6)). This nucleoprotein filament can pair the bound single strand with the complementary strand of an incoming duplex, resulting in homologous recombination.
The ssDNA binding protein from E. coli, SSB, affects formation of the nucleoprotein filament in several ways. In vitro, under standard reaction conditions that generally include 8–12 mM Mg2+, SSB stimulates filament formation on ssDNA substrates derived from bacteriophages by binding to and denaturing regions of secondary structure in the ssDNA that would otherwise hinder RecA filament extension (7). SSB is then displaced by the growing RecA filament (8). SSB thus permits the formation of a contiguous extended filament on the DNA. However, RecA and SSB bind ssDNA competitively in vitro, such that when SSB is prebound to ssDNA, it inhibits the nucleation stage of RecA protein filament formation (9,10). Subsequent binding of RecA to ssDNA results either when SSB transiently vacates a region of ssDNA, or when the RecOR mediator proteins facilitate RecA nucleation onto SSB-coated ssDNA (5,11,12).
Genetic studies also indicate that SSB inhibits RecA filament formation and subsequent homologous recombination. SSB protein has multiple DNA binding modes, and interconversion between them is mediated by salt concentrations (29–31). Mutations in recF, recO or recR genes, which belong to the same epistasis group, result in defects in the repair of stalled replication forks (13–18). These defects are likely due at least in part to the inability of RecA protein to displace SSB from the single-stranded region of the stalled fork. In support of this model, overproduction of SSB leads to a sensitivity to UV-inflicted DNA damage that is similar to the phenotype of recF mutants (19). Additional evidence supports a competition between RecA and SSB for ssDNA in vivo. The recF, recO or recR phenotypes can be partially suppressed, and the suppressor mutations map to the recA gene. These recA mutants include recA803 (V37M) (20, the contents of which is incorporated by reference here in its entirety), recA2020 (T1211) (21 and 22, the contents of both is incorporated by reference), recA441 (E38K+I298V) (23–25, the contents of each is incorporated by reference), and recA730 (E38K) (26 and 27, the contents of both is incorporated by reference). These RecA mutant proteins would need to be able to compensate in some way for the loss of the RecFOR proteins, and indeed RecA803, RecA441, and RecA E38K proteins all exhibit an enhanced ability to compete with SSB in vitro (8 and 28, the contents of both is incorporated by reference). RecA E38K protein competes best with SSB, followed by RecA441 and then RecA803 (8, the contents of which is incorporated by reference here in its entirety). However, these RecA mutants do not effectively displace SSB from linear single stranded DNA.
Indeed, Benedict and Kowalczykowski describe a study of a proteolytic fragment of RecA protein missing about 15% of the protein, which turned up in one purification prep of RecA protein. Although, some improved binding to duplex DNA was noted, the exact nature of the C-terminal deletion, greater than 25 amino acids, was never determined. Furthermore, no group has been able to reproduce the results since they did not report how many amino acids were deleted (37).
Likewise, Tateishi, et al. (38, the contents of which is incorporated by reference), describe a study of a purified RecA protein without 25 amino acids from the C-terminus. The authors also note some improved DNA binding. However, it has been found in work elaborated below that the described protein is still much less stable than RecA deletions desired for a variety of biological applications.
Also, Layery and Kowalczykowski describe a study of the E38K mutant alone (called RecA730). The authors note some improved displacement of SSB using the RecA730. However, for many applications, their results still indicate a substantial lag in the binding to DNA and displacement of SSB by RecA730.
Larminat and Defais, also studied a carboxyl terminal deletion of 17 amino acids in vivo. However, the mutant was never actually isolated and characterized as a pure protein (41). Furthermore, Yu and Egelman, disclose use of a pure RecA mutant with an 18 amino acid deletion. However, the study was confined to the structure of the resulting filaments. Thus, in neither of these two papers was the biochemistry of these proteins actually explored (42).
Thus, a RecA mutant protein is desired which can catalyze homologous DNA pairing and strand exchange reactions more efficiently due to an enhanced capacity of the mutant protein to compete with SSB and to provide a more persistent binding of the mutant protein to DNA.